1. Field of the Invention
This invention is directed to a method of diagnosing a tumor site in an individual by a non-invasive technique, and treating such tumor by a similar non-invasive technique.
2. Description of the Background Art
Currently available non-invasive diagnostic methods of tumor localization include isotope scans (e.g. liver scan, gallium scan), radiology (e.g. plain X-ray, barium meal, computerized tomography), and ultrasonography. Such methods vary in their sensitivity, depending upon the size, site, and histological type of cancer, but all of them are nonspecific. Zalcberg, J. R., Am. J. Clin. Oncol. 8:481-9 (1985).
Antibodies have long been recognized as potential target-specific imaging agents. Pressman et al. (J. Immunol. 59:141-6 (1948)) were the first to demonstrate conclusively the localization of radiolabeled antibodies in specific target organs in vivo. Since then, antibodies have been used for detection and visualization of various malignant tissues in experimental animals. (For a review, see Zalcberg, supra, and Carrasquillo, J. A., et al., Cancer Treatment Reports 68:317-28 (1984).)
Early studies used the immunoglobulin fractions of tumor-specific antisera for detection of tumors, or non-specific markers such as anti-fibrinogen, usually detectably labeled with radioactive 131.sub.I. More recently, antibodies to oncofetal proteins, also labeled with .sup.131 I, were used as immunologic tumor imaging agents; however, the amount of radiolabeled antibody localized in the tumor was low compared with that localized in the blood and other organs, particularly the liver, thus limiting the clinical utility of such methods.
Advances in the production of tumor-specific or tumor-associated antibodies led to the advent of monoclonal antibody technology to provide a source of large quantities of a specific antibody directed against a single epitope. Kohler, G., et al., Nature 256:495-7 (1975); Mach, J. R., et al., Immunol. Today 2:239-49 (1981).
Improvements in radiolabeling now permit the formation of stable, pharmacologically inert complexes of antibody with isotopes such as technitium-99 m or indium-111. These radiolabels, which have desirably short half-lives, allow high quality images to be recorded by scintigraphy with low radiation burden to the patient. Khaw, B. A., et al., J. Nucl. Med. 25:592-603 (1984).
Radiolabels have generally been attached to antibody proteins by two general techniques: oxidation methods, and coupling with cross-linkers. Oxidation methods include chloramine-T, lactoperoxidase, and chloramide iodogen (see, for example: Zalutzsky, M., et al., Int. J. Nucl. Med. Biol. 12 227-33 (1985); Sternthal, E., et al., New Engl. J. Med. 303:1083-8 (1980); and Marchalonis, J. J., et al., Biochem. J. 113:299--205 (1969)). Coupling of radiolabels to antibody proteins using cross-linkers such as diethylenetriaminepentacetic acid cyclic anhydride in the presence of SnCl.sub.2 and citrate in the presence of SnCl.sub.2 are the current methods of choice. (See, for example, Krejcarek, G. E., et al., Biochem. Biophvs. Res. Commun. 77:581-5 (I977); Khaw, B. A., et al., Science 209:205-7 (1980); Khaw, B. A., et al., J. Nucl. Med. 23:1011-19 (1982); Wong, D. W., U.S. Pat. No. 4,636,380; and Gansow, O. A., et al., U.S. Pat. No. 4,472,509.) Generally such labeling procedures produce a labeled immunoprotein which retains its physicobiological properties, is pharmacologically inert, and is suitable for imaging tumors.
Wong, U.S. patent supra, has disclosed the use of indium-111-labeled, tumor-specific autologous polyclonal antibodies for tumor imaging by scintigraphy. .sup.111 In-labeled human fibrinogen was also disclosed by this patent for neoplasm imaging.
Detectably labeled monoclonal antibodies directed to specific tumor antigens have achieved prominence in imaging specific tumors in vivo by scintigraphy (see, for example, Carrasquillo, supra: Khaw, supra; Zalcberg, supra; Nelp, W. B., et al., J. Nucl. Med. 28:34-41 (1987); Hayes, D. F., et al., Cancer Res. 46:3157-63 (1986); Murray, J. L., et al., J. Nucl. Med. 28:28-33 (1987); Larson, S. M., J. Nucl. Med. 26:538-45 (1985); Hwang, K. M., et al., J. Natl. Canc. Inst. 76:849-55 (1986); Goldenberg, M. D., U.S. Pat. No. 4,624,846; and Wong, D. W., U.S. Pat., supra).
Anti-tumor monoclonal antibodies (TMoAb) are directed against antigenic determinants that are selectively expressed on the surfaces of tumor cells. However, before such monoclonal antibodies can be used for imaging purposes, two essential criteria must be fulfilled. Hwang, supra. The first criterion is the target antigen specificity of the individual tumor monoclonal antibody. This is established by preliminary experiments on both tissue sections and cultured cells using a variety of techniques to establish both surface and Intracellular antigen expression. A second criterion, which also requires examination in a systematic manner, is the pharmacokinetics of the putative TMoAb; this involves a comparison of the uptake by tumor relative to that by normal tissue, determination of the extent of degradation of the antibody to inert species by the host's enzymes, and excretion of the antibody or fragments thereof by the kidney.
The preparation of a specific TMoAb that meets all necessary criteria for tumor imaging is a difficult, laborious and expensive process. Larson, supra. The overall procedure entails: (1) isolation and identification of the specific tumor antigen; (2) immunizing a mouse against this antigen; (3) preparing spleen cells from such an immunized mouse and fusing these cells with an immortal human cell line (e.g. myeloma cells); (4) establishing hybridoma colonies; (5) identifying hybridomas that secrete the antibody of interest, particularly those that produce large quantities; (6) cloning the hybridomas of interest; (7) isolating and purifying all of the different monoclonal antibodies; and (8) determining whether single or multiple TMoAb's are required for tumor imaging, as each monoclonal antibody is directed to a specific epitope on the tumor antigen.
Another complication in the use of TMoAb arises from its frequently inadequate concentration at tumor sites, and from its tendency to form immune complexes that are poorly excreted by the kidney. This, in turn, has created a need to use fragments derived from the monoclonal antibody for tumor imaging. It has been reported that antibody fragments such as F(ab').sub.2 or Fab, perhaps because of their relatively small size, diffuse more easily into tumors and are excreted more rapidly by the kidney. Thus, the tumor to blood ratio might be increased as a result of these two concurrent events. Zalcberg, supra at 484; Mach et al., supra Larson et al., supra: Khaw et al., supra: Carrasquillo et al., supra. The F(ab').sub.2 and Fab fragments of the immunoglobulin IgG represent the specific, variable N-terminal heavy and light chain domains of the immunoglobulin. These fragments must be prepared from the parent protein by proteolysis with specific proteinases, followed by isolation and rigorous purification.
Immunoglobulin molecules can bind to the surfaces of tumor cells by two mechanisms. The first requires the presence of a specific antigenic determinant on the cell surface, which interacts with an immunological site found in the variable region of the antibody. This region contains the tumor-specific F(ab).sub.2 and Fab fragments previously used in tumor imaging (see supra). The second mechanism requires a cell-surface receptor that binds to a non-specific constant (Fc) region of homologous and heterologous immunoglobulins. Such receptors are termed the Fc receptors. Various cells of the reticuloendothelial and lymphatic tissues (monocytes, macrophages, T and B lymphocytes), as well as malignancies from these cells (such as lymphoma, sarcomas, and leukemias), as well as certain types of breast and lung cancer, possess Fc receptors on their surfaces. The amino acid sequence of the heavy chain C-terminal domain of the immunoglobulin (Fc) fragment remains relatively stable, regardless of the antigenic stimulus.
Thus, there is a distinct difference between the preparation of an antibody that is capable of reacting specifically via its Fab region with a particular tumor-specific epitope, and a non-specific immunoglobulin that interacts with the Fc receptor on cells at the tumor site. A tumor imaging approach that can employ the latter mechanism would be highly desirable because of its simplicity and inexpensive nature.